Flavors and aromas, many of which originate from plants, have always had an important role in human culture. Ancient cultures cultivated and prized plants for their nutritional value as well as for their flavor, aroma and medicinal properties. However, the development of commercial large-scale agriculture in western civilizations resulted in emphasizing commercial/marketing interests in plant production, such as long shelf life, physical appearance and yield. The content of secondary metabolites, i.e. metabolites which do not have a defined metabolic role and presence of which is restricted to specific tissues, were often overlooked, although they significantly affect the nutritional value and aroma. Nowadays, there is growing awareness to healthy and flavorful plant products. Physically appealing but flavorless and aroma-less fruit is perceived as “synthetic”, while scented fruit is perceived as more “natural”. There is increasing public interest to “return” the natural flavor and aroma to fruits, emphasizing the importance of elucidating the relevant biosynthetic pathways, enzymes, genes and regulatory mechanisms involved. Cosmetic and food industries also seek natural aroma and flavors.
The monoterpene geraniol, which is emitted from flowers, notably roses (Rao et al 2000) and herbs (Mockute et al 1999) of many species, has an important role in their overall flavor and aroma. The mixture of geraniol's oxidation products, geranial and neral, also called citral, imparts a “lemon” flavor, and lemongrass (Singh Sangwan et al 1993), ginger (Miyazawa et al 1998), and some varieties of sweet basil (Grayer et al 1996) such as “Sweet Dani” are particularly rich in citral (Morales et al 1997). However, much is still lacking in the study of the physiological, biochemical and genetic regulation of the production of aroma compounds within the plants. Terpenoids are found in all plant species and have diverse physiological roles such as phytoalexins, pest deterrents and toxins, growth regulators, pollinator attractants, photosynthetic pigments and electron acceptors (Gershenzon and Croteau, 1993; Chappell, 1995; McGarvey and Croteau, 1995). For example, U.S. Pat. No. 6,258,602 discloses the isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint that produces the aphid alarm pheromone E-betafarnesene.
Monoterpenes are also one of the important groups of secondary metabolites involved in herb, fruit and flower aromas. While the backbones of the biosynthetic pathways leading to production of monoterpenes are ubiquitous to all plant species, the composition of terpenes often differs dramatically between species or even varieties leading to the diversity of aroma and flavors among herbs and fruits. This diversity seems to stem mainly from the specific composition and expression of the key-enzymes in the biosynthetic pathway, the terpene synthases, and additional downstream modification enzymes. Homology analysis revealed that while sequence conservation was not high among terpene synthases of different plant species, discrete conserved domains were present suggesting significant structural and functional similarity (Back and Chappell, 1995; Steele et al., 1998). These conserved domains have been the basis for isolation of a number of terpene synthases encoding genes from a variety of plant species using degenerate-primer based RT-PCR (Bohlmann et al., 1998a, 1998b, 1999; Steele et al., 1998).
Geraniol is likely to be synthesized from geranyl diphosphate (GDP), the universal precursor of all monoterpenes. Two types of enzymatic reactions have been hypothesized to lead to geraniol synthesis from GDP, either a phosphate-based or a monoterpene-based catalysis. However, in the absence of purified and characterized geraniol synthase, the question of whether GES employs a similar mechanism to the one used by other monoterpene synthases remained unsolved. This state of research limits the ability of the agricultural, food and cosmetic industries to use natural herb aromas, which are highly desirable in products of these industries.
Thus, there is a recognized need for, and it would be highly advantageous to have specific compounds involved in the regulation of herbal aroma pathways, and more advantageous to have isolated polynucleotides and enzymes capable of producing said aromas.